Melanoma is the most lethal form of skin cancer and is resistant to existing chemotherapeutic agents. Immunotherapy holds great promise in the treatment of metastatic melanoma because melanoma antigens have been identified and patients generate T and B cell responses specific for these antigens which in some cases lead to spontaneous remission. Effective immunotherapy requires presentation of tumor antigens in the context of MHC class II for the activation of CD4+ T lymphocytes to generate and sustain the anti-tumor immune response. Additionally, since many melanoma antigens represent self-antigens present in benign melanocytes, a productive anti-tumor response requires the avoidance of tolerance mechanisms which suppress the immune response to self-antigens. We propose to address the role of MHC class II antigen processing components, in particular gamma-interferon inducible lysosomal thiol reductase (GILT), in the immune recognition of melanoma. Melanoma antigens including tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2 and gplOO are likely to be substrates for lysosomal reduction by GILT, because these melanosomal membrane proteins are present in the MHC class II loading compartment and contain internal disulfide bonds. We have demonstrated that GILT is essential for the MHC class II processing of TRP-1 in vitro. In this proposal, we plan to explore the role of GILT in the development of tolerance to TRP-1 using a TRP-1-specific T cell receptor transgenic mouse model. We next plan to determine the role of GILT in the overall immune response to tyrosinase, TRP-1, TRP-2 and gplOO and to evaluate the role of GILT in protection from melanoma challenge by comparing in vivo immune responses in GILT-deficient compared to wild-type mouse strains. To explore the relevance in human disease, we will determine the expression of GILT in human melanoma. These studies will identify key features of antigen processing required to generate an immune response to melanoma and may aid in the understanding of mechanisms that lead to immune evasion. The long-term goal of these studies is to aid the development of effective immunotherapy for melanoma.